While several recent studies have reported on distinct ligand-dependent GPCR conformations, it remains unclear how these conformations elicit differential downstream responses. In contrast, deciphering G protein selection from functional response is complicated by the myriad of factors that regulate GPCR signaling, including relative abundance and availability of GPCR, G proteins, localization to different membrane surfaces or micro-domains, and the influence of regulatory proteins such as scaffolds, kinases, arrestins and the cellular endocytic apparatus. The proposed research addresses these limitations with a novel class of FRET-based SPASM sensors developed by the PI to systematically modulate protein-protein interactions in live cells. Research will focus o four GPCRs, b2-adrenoceptor (b2-AR), a2A-adrenoceptor, melanocortin4 receptor and b3-adrenoceptor, whose differential G protein selection has important functional consequences in the progression and treatment of heart failure, obesity and diabetes. Studies in aim 1 utilize sensors that detect the interaction between a GPCR and a peptide derived from the Ga subunit c-terminus, a known determinant of GPCR-G protein pairing. FRET-based measurements will be combined with established approaches to test the hypothesis that GPCRs adopt functionally distinct conformations in a ligand-dependent manner to trigger differential downstream responses. In preliminary studies, specificity of sensor measurements was tested with b2-AR and led to the identification of a functional Gi conformation triggered by the beta-blocker metoprolol. Studies are structured to dissect distinct events in the GPCR-G protein interaction followed by stimulation with canonical and biased agonists. A clear understanding of the GPCR-G protein interaction and G protein selection from FRET/BRET studies is limited by the lack of control over relative concentration. Studies in aim 2 will leverage the SPASM technique to delineate the sequence of events prior to and after ligand stimulation. Key questions addressed are: (1) is the ligand-free GPCR basally associated or pre-coupled to a G protein? (2) Does the GPCR dissociate from the G protein following ligand-stimulation? (3) Does G protein selection correlate with the inherent binding affinity of a GPCR for a G protein? (4) To what extent is biased signaling influenced by the local concentration of GPCR and G protein? Systematic variation of the concentration of the GPCR-G protein interaction using the SPASM technique will be used to assess the specificity of FRET and downstream responses. The proposed research has the potential to create a new technical and conceptual platform for future studies on ligand-driven signaling initiated by any GPCR. The extensive validation of the SPASM sensors is an integral part of this study, which in turn will pave the way for their use in live-cell drug screens to identify small molecules that bias GPCR signaling towards therapeutically desired outcomes.